The invention relates to systems and methods of culturing cells in organ assist devices.
Over 43,000 Americans die each year from liver disease, making it the tenth leading disease-related cause of death in the U.S. When liver disease progresses to liver failure, the mortality is 80% unless a compatible donor organ is found. As with other organs, there is a critical shortage of donor livers. Over 12,000 patients are currently listed as transplant candidates, but fewer than half that number of donor livers become available each year. Treatment with a liver assist device (LAD) would decrease the mortality associated with liver failure by stabilizing patients so that they are suitable candidates for a transplant, by supporting them until a suitable donor liver becomes available, and/or by preventing deterioration to the point where a liver transplant is required. Improving the pre-operative health of these patients would also increase transplant success, thereby decreasing the frequency of retransplantation and easing the demand for donor organs.
In cases of sudden or hepatic failure, which often occurs as a result of viral infection or toxicity, treatment with a LAD would eliminate the need for a transplant by supporting these individuals until their own livers regenerate. Liver transplantation is currently the most expensive organ transplant procedure. Successful development of a LAD would consequently provide major benefits to the US in reduced deaths and health-care costs.
Extracorporeal devices for temporary liver support have been investigated since the 1960s. Two strategies have been explored in the development of liver assist devices: (1) non-biological devices based on hemoperfusion on sorbents, hemodialysis across selectively-permeable membranes, and plasma exchange (Malchesky, xe2x80x9cNon-biological liver support: historic overview,xe2x80x9d Artif. Organs, 18:342-347, 1994); and (2) biological devices that incorporate cells or cellular components (Yarmush et al., xe2x80x9cAssessment of artificial liver support technology,xe2x80x9d Cell Trans., 1:323-341, 1992).
Non-biological devices have shown only limited efficacy, confirming that synthetic materials cannot replace the range and level of complex metabolic functions normally performed by the liver. On the other hand, a biological LAD in which hepatocytes are seeded on the outer surface of hollow fibers and blood or plasma circulates through the lumen of these fibers was proposed almost 25 years ago by Wolf and colleagues (Wolf et al., xe2x80x9cBilirubin conjugation by an artificial liver composed of cultured cells and synthetic capillaries,xe2x80x9d Tran. Amer. Soc. Artif. Int. Organs, 21:16-23, 1975).
Current biological LAD designs use the inverse of this concept today. Modern designs are often based on providing critical liver function by supporting high-density hepatocyte suspensions in hollow fibers, with circulation of blood or plasma outside the fibers. In this design, intermittent extracorporeal liver function is to be provided until the patient recovers through liver regeneration or until a transplant becomes available. However, the hollow fiber design is limited by several factors, including: a) inadequate mass transport, particularly of oxygen, b) lack of understanding of hepatocyte function in an in vitro environment, c) randomized tissue architecture for support of cell viability and function, and d) constraints of void volume on the perfusion circuit for the device.
Hollow fibers have been chosen for LADs on the basis of ready availability rather than demonstrated ability to support hepatocyte function. Perfusion of high-density hepatocyte cultures in hollow fibers has shown a lack of convincing benefit due to, among other reasons, transport limitations that undermine their support of high-density cultures. Such limitations are particularly acute for oxygen, which is required for both basic metabolic function as well as for initial steps in detoxification. Perfusion of oxygenated plasma or medium through or around a network of hollow fibers fails to address this problem because these aqueous liquids are poor carriers for oxygen and the associated distances for transport are relatively large. Modifications to the core hollow-fiber design (e.g., the use of a woven network of three independent sets of capillaries providing integral oxygenation) significantly complicate fabrication and incompletely address underlying transport limitations. They also lack the ability to orient hepatocytes in a more organotypic laminar configuration.
The invention features modular cell culturing devices comprised of one or more flat-plate modules. The invention is based on the discovery that if the flows of liquid medium and an oxygenated fluid are separated by a gas-permeable, liquid-impermeable membrane, and the cells are grown cultured on the liquid side of the membrane, the device can be used to culture cells with transport of oxygen through the membrane to the cells with independent control of the flow rate of the liquid passing through the device. The new flow-through cell culturing devices can thus be used to culture cells, e.g., hepatocytes, with high levels of cell function in organ, e.g., liver, assist systems, for production of cells, for production of cell-derived products, such as proteins or viruses, or for systems to treat biological liquids to remove toxins, such as ammonia, add cell-synthesized products, or both.
In general, the invention features methods and devices for the culture of cells that provide direct oxygenation of cells through planar, gas-permeable membranes. When the apparatus is seeded with the appropriate cells and is incorporated into a device, the device can be used to treat a patient with an organ, such as the liver, in need of functional assistance.
The invention features methods for culturing cells including: providing a gas-permeable, liquid-impermeable membrane having a first surface and a second surface; seeding cells on the first surface of the gas-permeable, liquid-impermeable membrane; contacting the cells with a nutrient-containing culture medium; providing an oxygenated fluid to the second surface of the gas-permeable, liquid-impermeable membrane at a pressure sufficient to provide transmembrane oxygenation to the cells seeded on the first surface; and culturing the cells under conditions that promote viability and function of the cells.
The device can be seeded with hepatocytes, e.g., porcine, equine, ovine, bovine, rabbit, rat, canine, feline, or murine hepatocytes. Additionally, the device can be seeded with human hepatocytes. The device can be seeded with 2 to 20 billion hepatocytes. The hepatocytes can be seeded directly onto the gas-permeable, liquid-impermeable membrane and then coated with collagen. Alternatively, the gas-permeable, liquid-impermeable membrane can be coated with collagen, and the hepatocytes can be seeded directly onto the collagen-coated membrane. Cells can seeded across the entire membrane from above the membrane.
In one embodiment, the oxygen contained in the oxygenated fluid is at or above the critical partial pressure of oxygen.
In one embodiment, the cells are preserved. The cells can be preserved by cryopreservation, hypothermic storage, or lyophilization.
The gas-permeable, liquid-impermeable membrane material can be made of, e.g., polystyrene, polyolefin, polyethylene, polypropylene, polyvinylidene fluoride, polycarbonate, hydrophobic-treated nylon, polyurethane, polyester, layered styrene-butadiene-styrene/ethyl vinyl acetate/styrene-butadiene-styrene, or layered styrene-butadiene-styrene/polyethylene.
The first surface of the gas-permeable, liquid-impermeable membrane can be treated, e.g., corona treated. In another embodiment, the first surface of the gas-permeable, liquid-impermeable membrane is collagen coated.
In one embodiment, the concentration of oxygen in the oxygenated fluid is between about 0% to about 90% oxygen. Additionally, the concentration of oxygen in the oxygenated fluid can be between about 19% to about 60%, or 40% to about 60%, oxygen. The concentration of oxygen in the oxygenated fluid can be controlled to promote or downregulate cell function.
In one embodiment, the nutrient-containing culture medium is perfused. Additionally, the method can further include filtering blood plasma.
The invention also features a flow-through cell culturing device including a housing with an oxygenated fluid inlet and an oxygenated fluid outlet, a liquid inlet and a liquid outlet, and first and second walls to form a chamber; a gas-permeable, liquid-impermeable membrane arranged between the first and second walls to separate the chamber into an oxygenated fluid compartment comprising an oxygenated fluid entry and an oxygenated fluid exit, and a liquid compartment comprising a liquid entry and liquid exit; and a liquid-permeable membrane arranged between a wall and the gas-permeable, liquid-impermeable membrane to separate the liquid compartment into a cell compartment and a liquid perfusion compartment, wherein the oxygenated fluid inlet and oxygenated fluid outlet are arranged such that oxygenated fluid entering the oxygenated fluid inlet flows into the oxygenated fluid entry and through the oxygenated fluid compartment and exits the oxygenated fluid compartment through the oxygenated fluid exit and the housing through the oxygenated fluid outlet, and wherein the liquid inlet and liquid outlet are arranged such that liquid entering the liquid inlet flows into the liquid entry and through the liquid-perfusion compartment and exits the liquid-perfusion compartment through the liquid exit and the housing through the liquid outlet.
In one embodiment, wherein in use, cells are seeded onto the gas-permeable, liquid-impermeable membrane, and the space between the gas-permeable, liquid-impermeable and liquid-permeable membranes is greater than the size of a cell. In addition, wherein in use, cells can be seeded onto either of the gas-permeable, liquid-impermeable membrane or the liquid-permeable membrane, and the space between the gas-permeable, liquid-impermeable and liquid-permeable membranes is about equal to the size of one cell. Additionally, wherein in use, cells can be seeded onto the gas-permeable, liquid-impermeable membrane, and onto the liquid-permeable membrane, and the space between the gas-permeable, liquid-impermeable and liquid permeable membranes is about equal to the size of two adjacent cells.
The device can further include a liquid-permeable hollow fiber arranged in the liquid compartment. Additionally, the housing can be arranged to enable stacking of one device on top of another device.
The invention also includes a liver assist system including a flow-through cell culturing device of the invention; a first conduit for conducting plasma from a patient to the housing inlet; a second conduit for conducting plasma from the cell culturing device to the patient; and a pump for moving plasma through the conduits and cell culturing device. The system can further include a plasma separator to remove blood cells from whole blood to provide plasma that is passed through the cell culturing device. The system can additionally include a bubble trap, to remove bubbles from the plasma in the first conduit prior to entering the cell culturing device.
The invention also features a liver assist system including a flow-through cell culturing device of the invention; an immunoisolation device; a first conduit for conducting plasma from a patient to an immunoisolation device; a second conduit for conducting plasma from the immunoisolation device to the patient; a third conduit for conducting liquid medium from the cell culturing device to the immunoisolation device; and, a fourth conduit for conducting liquid medium from the immunoisolation device to the patient; and, a pump for moving plasma through the conduits and cell culturing device.
The invention also includes a method of filtering blood plasma. This method includes seeding a flow-through cell culturing device of the invention with hepatocytes; introducing blood plasma into the liquid inlet of the device; supplying an oxygenated fluid into the oxygenated fluid inlet of the device; allowing the oxygenated fluid to flow through the oxygenated fluid compartment and out of the device through the oxygenated fluid outlet; and allowing the blood plasma to flow through the device and exit through the liquid outlet, thereby filtering the blood plasma.
The invention also includes a method for treating a patient in need of liver assist. The method includes attaching the liver assist system of the invention to the blood flow of a patient and treating the patient.
The invention also features a flow-through cell culturing device including a housing with a liquid inlet and a liquid outlet, an oxygenated fluid inlet and an oxygenated fluid outlet, and first and second walls to form a chamber; and a gas-permeable, liquid-impermeable membrane arranged between the walls to separate the chamber into an oxygenated fluid compartment comprising an oxygenated fluid entry and an oxygenated fluid exit, and a liquid compartment comprising a liquid entry and liquid exit, wherein the gas-permeable, liquid-impermeable membrane is seeded with cells, wherein the liquid inlet and liquid outlet are arranged such that biological liquid entering the liquid inlet flows into the liquid entry and through the liquid compartment and exits the liquid compartment through the liquid exit and the housing through the liquid outlet, and wherein the oxygenated fluid inlet and oxygenated fluid outlet are arranged such that oxygenated fluid entering the oxygenated fluid inlet flows into the oxygenated fluid entry and through the oxygenated fluid compartment and exits the oxygenated fluid compartment through the oxygenated fluid exit and the housing through the oxygenated fluid outlet.
The gas-permeable, liquid-impermeable membrane can be porous or non-porous. The gas-permeable, liquid-impermeable membrane comprises polystyrene, a polyolefin, polyethylene, polypropylene, polyvinylidene fluoride, polyurethane, poly(styrene-butadiene-styrene), poly(ethyl vinylacetate), nylon, silicon rubber, poly(tetrafluoroethylene), or composites, mixtures, or copolymers thereof. The gas-permeable, liquid-impermeable membrane can be surface treated, e.g., with a corona discharge or a coating of extracellular matrix.
In one embodiment, a gel is disposed on the cells. Alternatively, a gel can be disposed on the gas-permeable, liquid-impermeable membrane. The gel can contain cells suspended within said gel.
The invention also features a method of filtering blood plasma including seeding a flow-through cell culturing device of the invention with hepatocytes; introducing blood plasma into the liquid inlet of the device; supplying an oxygenated fluid into the oxygenated fluid inlet of the device; allowing the oxygenated fluid to flow through the oxygenated fluid compartment and out of the device through the oxygenated fluid outlet; and allowing the blood plasma to flow through the device and exit through the liquid outlet, thereby filtering the blood plasma.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The new flow-through cell culturing devices and organ assist systems provide numerous advantages. The new devices allow various cells to be cultured with desirable levels of mass transport of oxygen and other nutrients, waste products, and beneficial products, while potentially reducing detrimental shear stress normally associated with higher levels of media flow. As a result, even relatively shear-sensitive cells such as hepatocytes can be cultured for extended periods of time at relatively low media flow rates with high levels of function. As a consequence, oxygenation and perfusion can be controlled independently. Further, these devices allow direct treatment of surfaces for promotion of cell attachment and function as well as more uniform distribution of cells within the devices in the form of laminar cultures that simulate the in vivo architecture of the liver. These features allow the new flow-through cell culturing devices to be used in organ, e.g., liver assist systems.
Clinical studies have shown that adequate liver function can be maintained in vivo with as little as 10% of the normal cell mass suggesting that an effective LAD would support at least 1010 hepatocytes with approximately in vivo levels of function.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.